Instantaneous 4D micro-particle image velocimetry (µPIV) via multifocal microscopy (MUM).

Multifocal microscopy (MUM), a technique to capture multiple fields of view (FOVs) from distinct axial planes simultaneously and on one camera, was used to perform micro-particle image velocimetry (µPIV) to reconstruct velocity and shear stress fields imposed by a liquid flowing around a cell. A diffraction based multifocal relay was used to capture images from three different planes with 630 nm axial spacing from which the axial positions of the flow-tracing particles were calculated using the image sharpness metric. It was shown that MUM can achieve an accuracy on the calculated velocity of around (0.52 ± 0.19) µm/s. Using fixed cells, MUM imaged the flow perturbations at sub-cellular level, which showed characteristics similar to those observed in the literature. Using live cells as an exemplar, MUM observed the effect of changing cell morphology on the local flow during perfusion. Compared to standard confocal laser scanning microscope, MUM offers a clear advantage in acquisition speed for µPIV (over 300 times faster). This is an important characteristic for rapidly evolving biological systems where there is the necessity to monitor in real time entire volumes to correlate the sample responses to the external forces.

Peptide hydrogel in vitro non-inflammatory potential.

Peptide-based hydrogels have attracted significant interest in recent years as these soft, highly hydrated materials can be engineered to mimic the cell niche with significant potential applications in the biomedical field. Their potential use in vivo in particular is dependent on their biocompatibility, including their potential to cause an inflammatory response. In this work, we investigated in vitro the inflammatory potential of a ß-sheet forming peptide (FEFEFKFK; F: phenylalanine, E: glutamic acid; K: lysine) hydrogel by encapsulating murine monocytes within it (3D culture) and using the production of cytokines, IL-ß, IL-6 and TNFα, as markers of inflammatory response. No statistically significant release of cytokines in our test sample (media + gel + cells) was observed after 48 or 72 h of culture showing that our hydrogels do not incite a pro-inflammatory response in vitro. These results show the potential biocompatibility of these hydrogels and therefore their potential for in vivo use. The work also highlighted the difference in monocyte behaviour, proliferation and morphology changes when cultured in 2D vs. 3D. © 2016 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Dynamic loading of electrospun yarns guides mesenchymal stem cells towards a tendon lineage.

Alternative strategies are required when autograft tissue is not sufficient or available to reconstruct damaged tendons. Electrospun fibre yarns could provide such an alternative. This study investigates the seeding of human mesenchymal stem cells (hMSC) on electrospun yarns and their response when subjected to dynamic tensile loading. Cell seeded yarns sustained 3600 cycles per day for 21 days. Loaded yarns demonstrated a thickened cell layer around the scaffold׳s exterior compared to statically cultured yarns, which would suggest an increased rate of cell proliferation and/or matrix deposition, whilst maintaining a predominant uniaxial cell orientation. Tensile properties of cell-seeded yarns increased with time compared to acellular yarns. Loaded scaffolds demonstrated an up-regulation in several key tendon genes, including collagen Type I. This study demonstrates the support of hMSCs on electrospun yarns and their differentiation towards a tendon lineage when mechanically stimulated.

Polarization of porous hydroxyapatite scaffolds: influence on osteoblast cell proliferation and extracellular matrix production.

Improvements to clinically used biomaterials such as hydroxyapatite (HA) are of potential benefit to the patient. One modification, the addition of surface charges, has been shown to have an important role influencing cell response. In this study, porous HA scaffolds with both positive and negative surface charges were manufactured. The samples were sintered in air to produce porous HA ceramic scaffolds in the form of cylinders 12 mm in height × 7 mm in diameter. These were polarized with a dc voltage of 3 kV/cm. MC3T3E1 cells were placed on either negative or positive ends of the charged (or unpoled control) HA scaffolds. At 7 days, picogreen analysis was performed to analyze the cell number at the negative (4 mm), central (4 mm), and positive (4 mm) portions of the 12 mm cylindrical scaffold. At 4 weeks, micro-CT analysis was performed to quantify the regional volume of mineralized matrix deposition on the 3D scaffold. At 7 days, there were significantly more cells present at the negative end of the scaffold when seeded from the negative end in comparison to the other samples tested. Micro-CT data at 4 weeks correlated with this finding, demonstrating an increase in mineralized matrix at the negatively charged end of the scaffold seeded from the negative end in comparison to the positively charged and unpoled control scaffolds. The results indicate that the charge on HA influences cell activity and that this phenomenon can be translated to a clinically relevant porous scaffold structure.

In silico multi-scale model of transport and dynamic seeding in a bone tissue engineering perfusion bioreactor.

Computer simulations can potentially be used to design, predict, and inform properties for tissue engineering perfusion bioreactors. In this work, we investigate the flow properties that result from a particular poly-L-lactide porous scaffold and a particular choice of perfusion bioreactor vessel design used in bone tissue engineering. We also propose a model to investigate the dynamic seeding properties such as the homogeneity (or lack of) of the cellular distribution within the scaffold of the perfusion bioreactor: a pre-requisite for the subsequent successful uniform growth of a viable bone tissue engineered construct. Flows inside geometrically complex scaffolds have been investigated previously and results shown at these pore scales. Here, it is our aim to show accurately that through the use of modern high performance computers that the bioreactor device scale that encloses a scaffold can affect the flows and stresses within the pores throughout the scaffold which has implications for bioreactor design, control, and use. Central to this work is that the boundary conditions are derived from micro computed tomography scans of both a device chamber and scaffold in order to avoid generalizations and uncertainties. Dynamic seeding methods have also been shown to provide certain advantages over static seeding methods. We propose here a novel coupled model for dynamic seeding accounting for flow, species mass transport and cell advection-diffusion-attachment tuned for bone tissue engineering. The model highlights the timescale differences between different species suggesting that traditional homogeneous porous flow models of transport must be applied with caution to perfusion bioreactors. Our in silico data illustrate the extent to which these experiments have the potential to contribute to future design and development of large-scale bioreactors.

The composition of hydrogels for cartilage tissue engineering can influence glycosaminoglycan profile.

The injectable and hydrophilic nature of hydrogels makes them suitable candidates for cartilage tissue engineering. To date, a wide range of hydrogels have been proposed for articular cartilage regeneration but few studies have quantitatively compared chondrocyte behaviour and extracellular matrix (ECM) synthesis within the hydrogels. Herein we have examined the nature of ECM synthesis by chondrocytes seeded into four hydrogels formed by either temperature change, self-assembly or chemical cross-linking. Bovine articular cartilage chondrocytes were cultured for 14 days in Extracel, Pluronic F127 blended with Type II collagen, Puramatrix and Matrixhyal. The discriminatory and sensitive technique of fluorophore-assisted carbohydrate electrophoresis (FACE) was used to determine the fine detail of the glycosaminoglycans (GAG); hyaluronan and chondroitin sulphate. FACE analysis for chondroitin sulphate and hyaluronan profiles in Puramatrix closely matched that of native cartilage. For each hydrogel, DNA content, viability and morphology were assessed. Total collagen and total sulphated GAG production were measured and normalised to DNA content. Significant differences were found in total collagen synthesis. By day 14, Extracel and Puramatrix had significantly more total collagen than Matrixhyal (1.77+/-0.26 microg and 1.97+/-0.26 microg vs. 0.60+/-0.26 microg; p<0.05). sGAG synthesis occurred in all hydrogels but a significantly higher amount of sGAG was retained within Extracel at days 7 and 14 (p<0.05). In summary, we have shown that the biochemical and biophysical characteristics of each hydrogel directly or indirectly influenced ECM formation. A detailed understanding of the ECM in the development of engineered constructs is an important step in monitoring the success of cartilage regeneration strategies.

Polarization of hydroxyapatite: influence on osteoblast cell proliferation.

Hydroxyapatite (HA) has been used clinically to treat bone defects. However, modifications of the surface properties of HA could improve and control bone matrix deposition and localized host tissue integration. The aim of this study was to investigate the effect of developing a surface charge on HA discs with respect to osteoblast activity in vitro. HA discs (12 mm x 2 mm) were sintered in either air or water vapour. The HA discs were then electrically polarized (positive and negative surfaces) or non-polarized (controls) and seeded with MC3T3-E1 cells. Polarized HA sintered in water vapour was shown to retain six times more charge than polarized HA sintered in air. Picogreen analysis demonstrated that at 4h cell number was significantly higher on the negatively and positively charged HA surface (water sintered) in comparison to the non-charged water and air-sintered HA controls. At 7 days there was a significant increase in cell number on the negatively charged HA (air sintered) sample in comparison to the negatively charged water vapour sintered HA sample and the non-charged water vapour sintered control sample. Also at 7 days, the picogreen data showed a significant increase in cell number on the positively charged water-treated HA sample in comparison to both the air- and water-treated HA non-charged control HA samples. An alamarBlue assay at 7 days demonstrated significant cell metabolic activity on the charged surfaces (both positive and negative) in comparison to the non-charged HA and the tissue culture plastic controls. This study demonstrated that all of the HA discs tested supported cell viability/attachment. However, cell attachment/proliferation/metabolic activity was significantly increased as a result of developing a charge on the HA surface.

Bioreactors for bone tissue engineering.

Engineering bone tissue for use in orthopaedics poses multiple challenges. Providing the appropriate growth environment that will allow complex tissues such as bone to grow is one of these challenges. There are multiple design factors that must be considered in order to generate a functional tissue in vitro for replacement surgery in the clinic. Complex bioreactors have been designed that allow different stress regimes such as compressive, shear, and rotational forces to be applied to three-dimensional (3D) engineered constructs. This review addresses these considerations and outlines the types of bioreactor that have been developed and are currently in use.

Ectopic bone formation in bone marrow stem cell seeded calcium phosphate scaffolds as compared to autograft and (cell seeded) allograft.

Improvements to current therapeutic strategies are needed for the treatment of skeletal defects. Bone tissue engineering offers potential advantages to these strategies. In this study, ectopic bone formation in a range of scaffolds was assessed. Vital autograft and devitalised allograft served as controls and the experimental groups comprised autologous bone marrow derived stem cell seeded allograft, biphasic calcium phosphate (BCP) and tricalcium phosphate (TCP), respectively. All implants were implanted in the back muscle of adult Dutch milk goats for 12 weeks. Micro-computed tomography (microCT) analysis and histomorphometry was performed to evaluate and quantify ectopic bone formation. In good agreement, both microCT and histomorphometric analysis demonstrated a significant increase in bone formation by cell-seeded calcium phosphate scaffolds as compared to the autograft, allograft and cell-seeded allograft implants. An extensive resorption of the autograft, allograft and cell-seeded allograft implants was observed by histology and confirmed by histomorphometry. Cell-seeded TCP implants also showed distinct signs of degradation with histomorphometry and microCT , while the degradation of the cell-seeded BCP implants was negligible. These results indicate that cell-seeded calcium phosphate scaffolds are superior to autograft, allograft or cell-seeded allograft in terms of bone formation at ectopic implantation sites. In addition, the usefulness of microCT for the efficient and non-destructive analysis of mineralised bone and calcium phosphate scaffold was demonstrated.

Optimization of cell seeding efficiencies on a three-dimensional gelatin scaffold for bone tissue engineering.

Bone tissue engineering techniques hold great potential for the treatment of clinical defects. However, there is much optimization needed before bone tissue engineering can be used therapeutically. This study evaluated various cell seeding methods onto a porous three-dimensional (3D) scaffold for bone tissue engineering optimization. MG63 human osteoblast-like cells were seeded onto a resorbable, porous gelatin sponge in different suspension volumes (50 microl and 5 ml), and culture conditions, (static, shaken, rolled, or rotatory bioreactor). The DNA of the cells in the scaffold, the media and the containers were quantitated separately to determine the cell number and location after 3 days of culture. The samples were stained with calcein and viewed using confocal microscopy to determine cell viability and location. Placing a small cell suspension (50 microl) directly onto the scaffold produced a significantly higher proportion of cells adhered to the scaffold than a larger cell suspension (5 ml). In all conditions except the rotatory bioreactor, the percentage of cells remaining on the scaffold after 3 days in a small seeding volume (63 +/- 22%) was significantly higher than the larger seeding volume (36 +/- 25%). In the case of the rotatory bioreactor, the opposite appeared to be true (39 +/- 9% small volume and 72 +/- 14% larger volume). It was important to keep the seeding dynamics of the cultivated tissue engineered construct consistent throughout the experiments to ensure reproducibility. For this scaffold type, cells applied in a small volume and cultured on a plate shaker at 120 rpm (giving 81 +/- 14% of cells adhered to the scaffold) for 3 days is recommended.

Haemocompatiblity of controlled release glass.

There are many medical applications which benefit from the use of soluble biomaterials, including the sustained release of drugs over a precise period of time, or temporary conduits for controlling nerve regrowth. We have manufactured a series of phosphate-based controlled release glasses (CRGs) in which the solubility could be controlled by varying the concentration of CaO and Na2O. Fibres of the CRG containing iron and cerium were placed into direct contact with human neutrophils and macrophages in tissue culture for 2.5 and 24 h respectively and the responses analysed by scanning electron microscopy (SEM) and confocal microscopy. The supernatants were analysed for the cytokine IL-1beta by enzyme-linked immunosorbent assay (ELISA). Disks of CRG of various compositions were placed in contact with whole blood for 30 min and platelet adhesion assessed by SEM. Activation of platelets, granulocytes and complement were quantified by ELISA for beta-thromboglobulin, elastase and iC3b. Intrinsic coagulation activation was measured by timing the clotting of recalcified plasma. Only the cerium fibre inhibited IL-1beta release from macrophages. No platelet adhesion was observed to any disk composition. Three compositions containing MgO inhibited plasma clotting and showed an insignificant level of complement activation. This study has demonstrated the development of a number of compositions of CRG, which have great potential in a wide variety of biomedical applications.

Soft tissue response to glycerol-suspended controlled-release glass particulate.

Vesicoureteral reflux and urinary incontinence have previously been treated by various means including the endoscopic delivery of injectable bulking materials such as silicone micro-implants, PTFE implants, glass particles, fat and bovine collagen. These first three materials do not degrade and collagen requires frequently repeated injections in order to sustain the restored continence provided. Vesicoureteric reflux in children usually resolves independently before the age of five. Correction is required before this, because treatment by prophylactic antibiotics is frequently unsuccessful in preventing breakthrough infection. The ideal material for injection should have large particles to avoid migration, inject easily and controllably, be non-toxic and dissolve over the period of time by which time the kidney will be mature. Three different controlled-release glass (CRG) granule compositions have been prepared by Giltech Ltd, and suspended in a suitable carrier medium (in this case glycerol). The degradable glasses, which have two different size ranges of 200-300 and <53 microm, and three different solution rates, were injected intramuscularly into the dorso-lumbar region of rats. Histological analysis of cryostat cut section after time periods of 2 d, 4 and 9 wk, and 6 mon has been performed. Histology sections were stained for neutrophils and macrophages using enzyme histochemistry. ED1 (monocytes and immature macrophages), ED2 (mature tissue macrophages), CD4 (helper/inducer T-lymphocytes and macrophages), CD8 (suppressor/cytotoxic T-lymphocytes), Interleukin-1beta, IL-2 (activated T-lymphocytes), Major Histocompatibility Complex (MHC) class II (activated macrophages and activated B-lymphocytes), alpha-beta (T-lymphocytes) and CD45RA (B lymphocytes) antibodies have been used to stain immunohistochemically each sample. This study demonstrates that particulate, degrading glass is stimulating an inflammatory response in soft tissue at time periods up to 6 mon. It should be noted that very small particulate, fast degrading glass is leading to tissue necrosis and should not be considered further for these applications. However, larger particulate, slower degrading materials are demonstrating effective potential for stress incontinence applications.